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A dsRNA-degrading nuclease(dsrnase2)limits RNAi efficiency in the Asian corn borer(Ostrinia furnacalis)

Insect Science 2021年第6期28卷 1677-1689页

作者： Yun-He Fan Hui-Fang Song Mureed Abbas Yan-Li Wang Tao Li En-Bo Ma Anastasia M.W.Cooper Kristopher Silver Kun Yan Zhu Jian-Zhen Zhang Institute of Applied Biology Shanxi UniversityTaiyuanChina College of Life Science Shanxi UniversityTaiyuanChina Faculty of Biological Science and Technology Changzhi UniversityChangzhiShanxiChina Department of Entomology Kansas State UniversityManhattanKansasUSA

The efficiency of RNA interference(RNAi)varies substantially among different insect *** degradation of double-stranded RNA(dsRNA)by dsRNA-degrading nucleases(dsrnases)has been implicated to cause low RNAi efficiency in several insect *** this study,we identified four dsrnase genes(OfdsrnaseL Ofd-sRNase2,Ofdsrnase3 and Ofdsrnase4)from the Asian corn borer(Ostrinia furnacalis)transcriptome *** analyses showed that each deduced protein sequence contained endonuclease NS domains and signal *** expression analysis revealed that Ofdsrnase2 was exclusively expressed in the midgut of *** efficiency was investigated in 2-d-old fifth-instar larvae(high expression of dsrnase2)and 2-d-old pupae(low expression of dsrnase2)by feeding or injecting dsRNA targeting a marker gene that encodes the lethal giant larvae protein(OfLgl).Our results showed that OfLgl only partially silenced the expression of OfLgl in pupae,but not in larvae,suggesting that Ofdsrnase2 could contribute to lower RNAi efficiency in larval *** hypothesis was supported by our RNAi-of-RNAi experiment using a tissue culture technique where the silencing efficiency against the reporter gene,OfHexl,was significantly improved after knockdown of *** double luciferase assays were performed to evaluate the role of the four dsrnases in vitro,only Ofdsrnase2 expressed in S2 cells significantly affected RNAi efficiency by degrading *** together,our results suggested that the degradation of dsRNA by Ofdsrnase2 in the midgut contributed to low RNAi efficiency in *** larvae.

关键词： dsrnase Ostrinia furnacalis RNA interference RNAi efficiency tissue culture

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沉默茄二十八星瓢虫的dsrnase影响质体介导RNAi抗虫效率的研究

沉默茄二十八星瓢虫的dsRNase影响质体介导RNAi抗虫效率的研究

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作者：黎烊村湖北大学

学位级别：硕士

外源的双链RNA(double-stranded RNA,dsRNA)由害虫摄入后,会通过si RNA(small interfering RNA,si RNA)途径诱发RNA干扰效应(RNA interference,RNAi),害虫靶标基因表达下调从而导致生长发育迟缓甚至死亡,进而有效地保护了农作物。近年来,随着质体(叶绿体)转化技术的兴起,在植物质体中表达靶标害虫关键基因的dsRNA从而介导害虫的RNAi(plastid mediated-RNAi,PM-RNAi)以达到抗虫目的成为了研究热点。在质体转化马铃薯植株中表达靶标茄二十八星瓢虫(Henosepilachna vigintioctopunctata)关键基因的dsRNA,将该转基因植物喂食茄二十八星瓢虫后可对其二龄幼虫造成100%的死亡率。但茄二十八星瓢虫末龄幼虫对质体转化植株的抗性高于初龄幼虫。害虫体内的dsrnase(double-stranded ribonuclease)会降解dsRNA,从而导致RNAi抗虫效率降低,这也许是茄二十八星瓢虫末龄幼虫对PM-RNAi相对不敏感的原因之一。采用RNAi的方式将dsrnase敲低后,可提高具有低或中等抗虫效率的质体转化马铃薯植株对马铃薯甲虫的抗性,茄二十八星瓢虫和马铃薯甲虫同属鞘翅目,茄二十八星瓢虫体内是否也存在这种特殊的核酸酶,如果存在,那么沉默dsrnase后,是否也可以提高质体转化植株的RNAi抗虫效率成为了本研究的主题。针对以上问题,本研究开展了以下实验,实验内容及结果如下:1.筛选茄二十八星瓢虫的RNAi靶标基因。选取可溶性NSF附着蛋白基因(soluble NSF-attachment proteins,Snap)和β-肌动蛋白基因(β-actin)作为靶标,体外合成dsRNA,以ds Gfp(dsRNA specific to green fluorescent protein)和H2O为对照,分别喂食二龄幼虫,结果发现两个靶标基因均对能有效防治茄二十八星瓢虫,其中抗虫效率为ds Actin>ds Snap。2.构建分别表达ds Actin和ds Snap的质体转化载体,通过基因枪轰击质体转化马铃薯。将同质化的质体转化马铃薯的叶片喂食二龄的茄二十八星瓢虫后发现,转基因植物对茄二十八星瓢虫的抗性与体外生测结果一致:St-ds Actin>St-ds Snap。前期研究表明沉默马铃薯甲虫的dsrnase后,仅对中低强度抗虫效果的质体转化植株具有提高其抗性的作用,因此选择了抗虫强度相对较弱的St-ds Snap开展后续实验。3.分析茄二十八星瓢虫的转录组数据。经鉴定,茄二十八星瓢虫体内存在两种dsrnase,分别命名为Hvdsrnase1和Hvdsrnase2,Hvdsrnase1和Hvdsrnase2分别在4龄幼虫和成虫中显著高表达,并且Hvdsrnase1和Hvdsrnase2均在幼虫的中肠中高表达。4.体外合成靶标这两种Hvdsrnase的dsRNA,为别是dsdsrnase1、dsdsrnase2、和同时靶标dsrnase1和dsrnase2的融合片段dsdsrnase1/2。为研究茄二十八星瓢虫取食dsdsrnase后dsrnase基因下调效率,将分别涂抹了dsdsrnase1、dsdsrnase2、dsdsrnase1+dsdsrnase2、dsdsrnase1/2的马铃薯叶片喂食茄二十八星瓢虫,在第2、3和4天取样并分析样品dsrnase基因表达情况后发现,dsdsrnase1+dsdsrnase2、dsdsrnase1/2处理2天后,均能同时显著下调Hvdsrnase1和Hvdsrnase2两个基因的表达。***-of-RNAi的实验。将dsdsrnase1/2涂抹于St-ds Snap叶片,以野生型马铃薯(Stwt)涂抹H2O为对照。喂食三龄初的茄二十八星瓢虫,实验结果表明,St-wt+dsdsrnase1/2与St-wt+H2O两组试虫之间的存活率无显著变化,均高于St-ds Snap+dsdsrnase1/2与Stds Snap+H2O;St-ds Snap+dsdsrnase1/2组的体重、取食面积、化蛹率以及幼虫的存活率均显著低于St-ds Snap+H2O,且St-ds Snap+H2O组在第7天发生“跃迁式”的死亡(指害虫取食质体转化植物初期,死亡率并无显著变化,直到某个时间点,供试虫同一时间大量死亡),而St-ds Snap+dsdsrnase1/2“跃迁式”的死亡提前1天发生(第6天)。然而,Stds Snap+dsdsrnase1/2和St-ds Snap+H2O两组之间第5天的Snap基因的表达并无显著差异。综上所述,预先对茄二十八星瓢虫的Hvdsrnase基

关键词：茄二十八星瓢虫 dsrnase RNAi-of-RNAi 质体双链RNA

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Gene cloning,protein expression,and enzymatic characterization of a double-stranded RNA degrading enzyme in Apolygus lucorum

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Insect Science 2024年第1期31卷 119-133页

作者： Jie-Yu Zhang Jing Zhao Keyan Zhu-Salzman Qin-Qin Ji Yi-Ping Jiang Liu-Bin Xiao De-Jin Xu Guang-Chun Xu Lin-Quan Ge Yong-An Tan College of Plant Protection Yangzhou UniversityYangzhouJiangsu ProvinceChina Institute of Plant Protection Jiangsu Academy of Agricultural SciencesNanjingJiangsu ProvinceChina Department of Entomology Texas A&M UniversityCollege StationTXUSA Taizhou Customs of the People's Republic of China TaizhouJiangsu ProvinceChina

RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic ***,silencing efficacy varies greatly among different insect ***,we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA *** disappearance of double-stranded RNA(dsRNA)could be a potential factor that restricts RNAi ***,we found that dsRNA can be degraded in midgut fluids,and a dsrnase of ***(Aldsrnase)was identified and *** alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects’*** signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali *** showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle,with peaks at the 4th instar ecdysis in the whole *** purified Aldsrnase protein obtained by heterologously expressed can rapidly degrade *** comparing the substrate specificity of Aldsrnase,3 specific substrates(dsRNA,small interfering RNA,and dsDNA)were all degraded,and the most efficient degradation is ***,immunofluorescence revealed that Aldsrnase was expressed in the cytoplasm of midgut *** cloning and functional study of Aldsrnase,the enzyme activity and substrate specificity of the recombinant protein,as well as the subcellular localization of nuclease,the reason for the disappearance of dsRNA was explained,which was useful in improving RNAi efficiency in *** and related species.

关键词： Apolygus lucorum cloning dsrnase enzymatic activity protein expression

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